Maintaining the fidelity of a nucleic acid sequence is of paramount importance in many aspects of molecular biology. During the cloning of a gene sequence, for example, the cloning processes themselves can introduce artifacts into the sequence being cloned, resulting in the isolation of aartificial variant sequence, rather than the true original sequence.
For instance, PCR amplification is known to create mutations at a much higher rate than in vivo propagation (replication) of DNA. The mutation rate will vary with PCR conditions, choice of enzyme, number of replication cycles, etc. After 30 rounds of PCR amplification, it is estimated that between 2% and 10% of the molecules (depending on their size) contain at least one mutation. Thus, when PCR amplified DNA is cloned into a plasmid vector, 2–10% of the cloned inserts will contain mutations. Depending on the downstream application, it is often necessary to sequence the amplified DNA prior to further experimentation. However, since many cloning procedures involve some amplification steps, sequencing can only show if additional variations have been introduced, and cannot demonstrate that the current sequence is true to the original.
In another instance, when assaying sequences within samples for the presence of alterations that indicate the possibility of genetic disease, it becomes critical to know that one is working with the actual sequence originally sampled, rather than an amplification-induced artifact.
In studying variations between the genomes of related organisms and populations, it is also critical to know that one is studying actual sequence differences instead of artifacts. Some assays of genetic diversity within and between human populations, for example, test for the presence of single nucleotide polymorphisms (SNP). Variations in sequences that are actually due to amplification can produce in spurious results.
There is therefore a need in the art for methods that select for PCR amplification products and other nucleic acids that do not contain PCR-induced mutations or other alterations.